Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Testing the effects of nitric oxide (oxidative) stress on Caulobacter transcription

ABSTRACT: The goal of this study was to measure the effects of nitric oxide exposure (using DETA NONOate as a nitric oxide donor) on transcription in Caulobacter. Untreated Caulobacter crescentus were grown to a density of 0.3 (at OD660) in PYE medium (pH 7) in rolled culture tubes. DETA-NONOate treated Caulobacter crescentus were grown to a density of 0.3 (at OD660), and then treated with 100 mM DETA NONOate for 30 minutes.

ORGANISM(S): Caulobacter vibrioides

SUBMITTER: Sean Crosson

PROVIDER: E-GEOD-21139 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Interaction specificity, toxicity and regulation of a paralogous set of ParE/RelE-family toxin-antitoxin systems.

Fiebig Aretha A Castro Rojas Cyd Marie CM Siegal-Gaskins Dan D Crosson Sean S

Molecular microbiology 20100512 1

Toxin-antitoxin (TA) gene cassettes are widely distributed across bacteria, archaea and bacteriophage. The chromosome of the alpha-proteobacterium, Caulobacter crescentus, encodes eight ParE/RelE-superfamily toxins that are organized into operons with their cognate antitoxins. A systematic genetic analysis of these parDE and relBE TA operons demonstrates that seven encode functional toxins. The one exception highlights an example of a non-functional toxin pseudogene. Chromosomally encoded ParD a ...[more]

PMID: 20487277

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Project description:Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Consequently, cells in all domains of life have evolved mechanisms to repair DSBs, including through homologous recombination. Although recombination has been well characterized, the spatial organization of this process in living cells remains poorly understood. Here, we introduced site-specific DSBs in Caulobacter crescentus, and then used time-lapse microscopy to visualize the homology search, DSB repair, and the resegregation of chromosomal DNA. Even loci tethered to opposite cell poles can efficiently release, pair to enable recombination-based repair, and then resegregate to their original locations. Resegregation occurs independent of DNA replication and without disrupting global chromosome organization. Origin-proximal regions are resegregated by the same machinery, ParABS, used to segregate undamaged chromosomes following DNA replication. In contrast, origin-distal regions efficiently resegregate after a DSB independent of ParABS, and likely without dedicated segregation proteins. Instead, we propose that a physical, spring-like force drives the resegregation of origin-distal loci after DSB repair. Caulobacter cells were depleted of DnaA for 1.5 h before synchronization. Swarmer cells were then released into DnaA depleting conditions (without IPTG) and double-strand breaks were induced for 1 h by the addition of 500 ?M vanillate. For control sample, no vanillate was added. Formadehyde (Sigma) was then added to the final concentration of 1%. Formadehyde crosslinks protein-DNA and DNA-DNA together, thereby capturing the structure of the chromosome at the time of fixation. Fixation was performed at the cell density of OD600 = 0.2. The crosslinking reactions were allowed to proceed for 30 minutes at 25 °C before quenching with 2.5 M glycine at a final concentration of 0.125 M. Fixed cells were then pelleted by centrifugation and subsequently washed twice with 1x M2 buffer (6.1 mM Na2HPO4, 3.9 mM KH2PO4, 9.3 mM NH4Cl, 0.5 mM MgSO4, 10 ?M FeSO4, 0.5 mM CaCl2) before resuspending in 1x TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA) to a final concentration of 107 cells per µl. Resuspended cells were then divided into 25 µl aliquots and stored at -80 °C for no more than 2 weeks. Each Hi-C experiment was performed using two of the 25 µL aliquots. Chromosome conformation capture with next-generation seqeuncing (Hi-C) was carried out exactly as described previously (Le et al., 2013 PMID: 24158908)

Dataset Information

Testing the effects of nitric oxide (oxidative) stress on Caulobacter transcription

Publications

Interaction specificity, toxicity and regulation of a paralogous set of ParE/RelE-family toxin-antitoxin systems.

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